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E llingson et al .: J ournal of AOAC I nternational V ol . 99, N o . 1, 2016  207

Table 2. Choline precision summary a

Free choline

Total choline

Mean concn, mg/100 g

,

RSD INT , %

Mean concn, mg/100 g

,

RSD INT , %

RSD r

RSD r

%

%

Sample

81.3

2.1 2.3

105 2.1 1.8

SRM

1849a b,c

0.364 d

1.8 d,e

2.7 d,f

4.11 2.3 g

2.8 f

AN powder milk protein- based IF hydrolyzed milk-based IF hydrolyzed soy-based AN powder low fat Child formula powder IF elemental powder

14.4

1.7 2.7

18.8 2.1 3.5

17.8

1.7 2.7

17.8 2.0 2.9

18.1 2.4 h

2.8

15.5

1.6 3.5

4.61

1.0 1.7

5.70 2.3 2.6

Figure 2015.10B. Extracted ion chromatogram (XIC) of carnitine.

Table 2015.10B. Compound transitions

8.11

1.1 2.2

8.68 2.2 2.8

Transition (Q1/Q3)

Collision energy, V

Retention time, min

17.4 2.1 i

3.3 f

Compound

Use/type

IF powder

13.8

2.2 2.5

milk-based

Carnitine

Quantitation 162.0/103.0

25 25

1.7 1.7

IF powder

14.9

1.1 2.1

20.2 2.6 4.0

Carnitine-d3 Internal standard

165.0/103.0

soy-based

12.6 3.1 i

2.7 g

21.4 2.8 2.5

IF RTF milk- based AN RTF high protein

Carnitine Carnitine Choline

Confirmation 162.0/84.4 Confirmation 162.0/59.1 Quantitation 104.2/60.0

29 27 25 25

1.7 1.7 2.1 2.1

42.8

2.7 2.6

49.3 1.6 1.5

Choline-d4

Internal standard

108.2/60.0

AN RTF high fat

48.1

1.4 1.5

53.2 1.2 1.5

Choline

Confirmation 104.2/45.2

25

2.1

0.205 d

2.2 d

4.0 d

1.25 d

2.7 d

2.2 d

Child formula powder placebo IF elemental powder placebo AN RTF high protein placebo AN RTF high fat placebo

where R = results expressed in mg/100 g, C = concentration of the analyte in the injected solution in ng/mL, V = volume of the initial extract in mL, S = sample weight in grams, and D = dilution factor, the inverse of any dilution made. All results were calculated on a RTF or reconstituted basis of 25 g diluted to 225 g with water, except for SRM 1849a that was calculated back to powder basis.

0.118 d

6.4 d

6.4 d

0.651 d

1.8 d

3.0 d

0.491 d

1.7 d

1.9 d

6.03 3.4 3.2

0.522 d

2.8 d

2.8 d

5.10 2.7 2.5

Results and Discussion

IF RTF

5.42

1.6 2.4

13.2 2.0 2.2

Linearity was assessed from the percent deviation from the theoretical concentration across the working standard range (Table 1). The results from the validation showed an overall repeatability for free and total choline of 1.9 and 2.3% RSD r , whereas the overall intermediate precision obtained for free and total choline was 2.4 and 2.7% RSD INT , respectively (Table 2). Free and total carnitine had an overall repeatability of 2.9 and 2.7% RSD r , whereas the overall intermediate precision obtained for free and total carnitine was 3.3 and 3.1% RSD INT ,

milk-based placebo

Overall 2.3 2.7 a  Samples were run in triplicate across 4 days, n = 12. Deviation is footnoted due to outliers by Grubbs’ test at a 95% confidence interval, loss of sample during preparation, or failure to prepare in triplicate. Twelve data points were still obtained for all samples. b  Free choline information mass fraction value of 79.8 mg/100 g. c  Total choline certified mass fraction mean and range of 109 mg/100 g and 98.0–120 mg/100 g, respectively. d  Results below the SMPR-required LOQ of 2.0 mg/100 g and are not included in the calculations. e  Three days of duplicates used for calculation. f   Calculated across 5 days. g  Two days of duplicates and 1 day of four replicates used for calculation. h  One day of duplicates and 1 day of four replicates used for calculation. i   One day of duplicates used for calculation. 1.9 2.4

Table 1. Linearity a

Percent deviation from theoretical concn, ng/mL

Compound 10

20

500 2000 4000 5000 Mean 2.0 2.4 1.2 2.0 2.2

Carnitine 2.9 3.0

Choline 1.4 1.1 1.0 1.7 1.9 a  Average from three analyses of duplicates at each working standard level. 2.5 4.1